Penicillin aldehydes



United States Patent "Ce 3,316,273

Patented Apr. 25, 1967 3 316 273 Preferred compounds of the presentinvention are those of the formulae PENICILLlN ALDEHYDES William J.Gottstein and Lee c. Cheney, Fayetteville,

N.Y., assignors to Bristol-Myers Company, New York, 5 0 c c -c11 04311,H N.Y., a corporation of Delaware 5 l N0 Drawing. Filed Mar. 20, 1964,Ser. N0. 353,577

13 Claims. (Cl. 260306.7) This invention relates to novel antibacterialagents and f chemical reagents and, more particularly, to 6-substitutedArXC-C-NHOHOH C CH3 H penicillanyl aldehydes and especially topenicillin alde- LW L Q hydes.

The aldehydes of the present invention are effective antibacterialagents in vivo against Gram-positive bacteria 3 and are also effectiveagents for use in a search for oXida- 15 Z H tive enzymes.

There is thus provided, according to the present invention, the seriesof compounds having the formula wherein R is amino, acylamino,benzyloxycarbonylamino, ph-thalimido or tritylamino. In these compoundsthe Ar moiety may exist in tautomeric equilibrium with the enolicstructure 0 I R -H--( JNH-(IJHC \CZCHB H -C=(|3OH N i RD O=CN-OHC=O H\O/ The preferred series of compounds is that of the O formula t /s\/GH3 I 0 I s CH3 R2 Q NH JH 40 %NHCHO ofoH; 11 O=C -r N H7C=0 O=()-N-CH-C 0 in which R represents the side chain of any of the known X sCH penicillins other than those containing a group which is H 3 alteredby reaction with Raney nickel at 0 C. to 50 C. Ar-NHCNH(|3HCH ooH3 H asdetermined by simple test. It is this series which may be named 6-(substituted formamido)penicillanyl aldehydes or, more simply,penicillin aldehydes. S

In addition to the Well-knovvn, older penicillins pro- E Z H duced bythe use of precursors in fermentation processes, other known penicillinsinclude those disclosed in US. Patents 2,941,995, 2,951,839, 2,985,648,2,996,601, 3,007,- 920, 3,025,290, 3,028,379, 3,035,047, 3,040,032,3,040,033, 3,041,332, 3,041,333, 3,043,831, 3,053,831, 3,071,575, (C HCNHOH-CH 0-011 H 3,071,576, 3,079,305, 3,079,306, 3,080,356, 3,082,204,I, 3,093,547, 3,093,633, 3,117,119, 3,118,877, 3,120,512, 3,120,513,3,120,514; in British patent specifications 6 874,414, 874,416, 876,516,876,662, 877,120, 877,323, C 877,531, 878,233, 880,042, 880,400,882,335, 888,110, S 1 888,552, 889,066, 889,069, 889,070, 889,168,889,231, H 890,201, 891,174, 891,279, 891,586, 891,777, 891,938, I I893,518, 894,247, 894,457, 894,460, 896,072, 899,199, 0 900,666,902,703, 903,785, 904,576, 905,778, 906,383,

918,169, 920,176, 920,177, 920,300, 921,513, 922,278, 0 s CH3 924,037,925,281, 931,567, 932,644, 938,066, 938,321, g \CiCH: H 939,708,940,488, 943,608, 944,417; in numerous pub- 1 II I HAJZO lished Belgianpatents, e.g. 593,222, 595,171, 597,859, 602,494, 603,703, 609,039,616,419, 617,187; and South 1 0 S African patent applications, e.g.60/2882, 60/3057, 60/ l C i H 3748, 61/1649, R61/2751, 62/54, 62/4920,63/1612 and 63/2423. Za 0= NOHC=0 therein R represents hydrogen, amino,carbobenzoxymino, phenyl, fluoro, chloro, bromo, iodo, hydroxy, orlower)alkanoyloxy including especially acetoxy or (lowr)alkoxy; Xrepresents oxygen or sulfur; R and R each epresent hydrogen, phenyl,benzyl, phenet-hyl or (lowr)alkyl; R represents (lower)alkyl; R and Reach repesent (lower)alkyl, (lower)alkylthio, benzylthio, cyclo- 1 IIAr-C- and Ar represents the monovalent radical of the formula wherein RR and R are each a member selected from the group consisting ofhydrogen, chloro, bromo, iodo, trifluoromethyl, phenyl, (lower)alkyl and(lower)alkoxy, but only one R group may represent phenyl.

The term (lower)alkyl as used herein means both straight and branchedchain aliphatic hydrocarbon radicals having from one to ten carbon atomssuch as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,amyl, hexyl, 2-ethylhexyl, heptyl, decyl, etc. Similarly, where the term(lower) is used as part of the description of another group, e.g.,(l0wer) alkoxy, it refers to the alkyl portion of such group which istherefore as described above in connection with (lower)alkyl.

Preferred embodiments of the present invention are the restricted seriesof compounds of the following formulae wherein R represents (lower)alkyl;

wherein R represents (lower)alkyl and R and R each represent a memberselected from the group consisting of hydrogen and chloro;

wherein R is (lower)alkyl and R is a member selected I, from the groupconsisting of hydrogen and chloro; 0

o=o-N-- crr-o=o wherein R represents (lower)alkyl;

wherein R is (lower)alkyl; and the individual compounds of the followingformulae O-G-C'H O=C-N--CI-IC=O The compounds of the present inventionare prepared,- according to the present invention, by either of twomethods, as follows:

METHOD A.-OXIDATION OF PENICILLIN ALCOHOLS The present penicillinaldehydes are prepared by oxidation of the corresponding penicillinalcohols by the addition of about 2-5 moles of a carbodiimide (e.g.dicyclohexylcarbodiimide, diisopropylcarbodiimide) to a solution ofabout one mole of the alcohol and 0.1-2.0 mole anhydrous phosphoric acidin dry dimethyl sulfoxide. Tetramethylene sulfoxide may also be used andthe sulfoxide may be diluted with up to nine volumes of an inertsolvent. The reaction proceeds readily at room temperature. Thephosphoric acid functions as a catalyst and may be replaced withphosphorous acid, cyanoacetic acid or pyridinium phosphate,trifluoroacetate, hydrochloride or sulfate. Further details are given inthe examples below.

METHOD B.--HYDROGENOLYYSIS BY RA'NEY NICKEL OF PENICILLIN THIOACIDS ANDTHIOESTERS The penicillin aldehydes of the present invention arepreferably prepared by hydrogenolysis by Raney nickel of athiopenicillin (also called a penicillin thioacid; e.g. see US. Patent2,751,378) which is preferably in the form of the free acid or of a saltsuch as an alkali metal salt or an amine salt. The reaction is carriedout at 50 C. and preferably at close to 0 C. It is advantageous to usean anhydrous or nearly anhydrous solvent such as tetrahydrofuran oracetone and to use catalytic amounts of a weak anhydrous acid such asglacial acetic acid as the penicillin aldehydes are very sensitive tobases. It is advantageous to add sufficient water to dissolve thethiopenicillin in the reaction mixture in the event that it is notcompletely soluble in the anhydrous solvent, The proportions are notcritical but it is of course desirable to use enough Raney nickel toassure a high yield in the reaction; it is thus preferred to use aweight of Raney nickel (on a Wet basis) equal to at least three timesthe weight of the thiopenicillin. The activated Raney nickel availablecommercially is quite suitable for use as is that prepared according toMozingo et al., J. Amer. Chem. Soc., 65, 1013 (1943). Thethiopenicillins can be replaced, if desired, with their esters, e.g.with alkylthio or aralkylthio esters; see J. Amer. Chem. Soc., 75,3636-3637 (1953), J. Chem. Soc. (London), 3733-3739 (1953) and Chem.Ber., 92, 530-534 (1959).

It is on occasion advantageous, but by no means essential, to conductthe hydrogenolysis in the presence of a trapping agent for the aldehydebeing produced. By this is meant an N,N'-disubstituted-ethylenediamineor a 1,3-disubstituted-propane-1,3-diarnine. These reagents condensewith the aldehyde formed during the course of the hydrogenolysis to formthe corresponding imidazo lidines and hexahydropyrimidines respectively,thus protecting the aldehyde function from further reduction or otherreaction, The penicillin aldehyde can be readily regenerated bytreatment of these heterocyclic derivatives with an acid, e.g., withp-toluenesulfonic acid monohydrate in a suitable solvent such asacetone-ether.

Examples of preferred diamines for use as the aldehyde trapping agentsare those of US. Patent 2,739,981 and especiallyN,N'-dibenzylethylenediamine [W. F. Minor, D. A. Johnson and L. C.Cheney, J. Org. Chem., 21, 528 (1956)], N,N-diphenylethylenediamine [W.Wanzlick and W. Lochel, Chem. Ber., 86, 1463 (1953)]. Other suitablediamines include N,N'-dimethy1ethylenediamine [A. J. Birch, J. CymermanCraig and M. Slaytor, Australian J. Chem, 8, 512 (1955)] and N,N-bis-(p-methoxybenzyl)-1,3-diaminopropane [J. H. Billman and J. L.Meisenheimer, J. Med. Chem., 6, 682 (1963)].

The product is easily separated from the Raney nickel by filtration andfrom unreacted starting thiopenicillin by virtue of the fact that onlythe latter contains an acidic group and can thus be extracted from anorganic solvent, such as ether, into aqueous alkali, e.g. 5% NaI-ICOWhere desired, the penicillin aldehydes may be purified by reaction with2,4-dinitrophenylhydrazine or N,N-di'benzylethylenediarnine [of J. Org.Chem, 21, 528-529 (1956) and US. Patents 2,717,893 and 2,767,168] toform the hydrazone or imidazolidine respectively, which can itself bepurified as by recrystallization and then treated to regenerate purifiedpenicillin aldehyde, e.g. by acid hydrolysis or aldehyde interchange.

The compounds of the present invention exhibit in vitro antibacterialactivity. Thus when the Minimum Inhibitory Concentration (MIC) inmeg/ml. was determined in heart infusion broth (to which 5% pooled humanserum had been added) for the compounds having the structures Despitethese low levels of in vitro antibacterial activity, t was surprisinglydiscovered that these compounds are :ffective antibacterial agents invivo, that is, exhibited a ninirnum curative dose in 50% of the micetested (CD against an overwhelmingly lethal dose of S. aureus Smith isfollows:

Compound: CD in mgm./ kg. I 230 II 500 III 6.0 IV 3.4 Ampicillin 0.6

1 Complete protection (zero deaths) at 500 mgm./kg.

oxidative enzymes in the microorganism being examined.

The following examples are presented to illustrate the present inventionbut not to limit it. All temperatures are given in degrees centigrade.

EXAMPLE 1 O C H3 6-phenoxyacetamidopenicillanal. Commercial, pyrophoricRaney active nickel catalyst, 125 g. wet weight, was washed three timeswith 275 ml. portions of absolute ethanol and then with four 275 ml.portions of tetrahydrofuran (Tl-IF). The nickel was then suspended in500 ml. of THF, 10.5 ml. (0.175 mole) of glacial acetic acid was addedand the mixture was stirred and cooled to 4. A solution of 20.22 g.(0.0500 mole) of the potassium salt of penicillin V thiol acid wasprepared by suspending the solid in 200 ml. THF and adding 15 ml. Water.This solution was added to the suspension of nickel and the mixture wasstirred at 2 for 30 minutes. The catalyst was removed by filtrationthrough diatomaceous earth (Supercel) which was then washed with five150 ml. portions of THF. From the combined, reddish filtrates thesolvent was removed in vacuo at 33. The residue was taken up in 1700 ml.ether which was then extracted with three 250 ml. portions of 5% NaHCOsolution to remove unchanged starting material; the aqueous extractswere discarded. The ethereal phase was then washed with three 175 ml.portions of water, dried over sodium sulfate, decolorized with charcoaland filtered through Supercel. Complete removal by distillation of thesolvent from the filtrate left the product,6-phenoxyacetamidopenicillanal, as a stiff gum (5.7 g.). It wasconverted to a white solid by twice dissolving it in about 15 ml.methylene chloride which was then poured into 800 ml. n-pentane toprecipitate 4.0 g. of the product as a white solid.

EXAMPLE 2 O-CHs The compounds of the present invention are useful agentsfor the detection of microorganisms containing oxidative enzymes, e.g.of the type used to oxidize steroids. Thus, cells of the microorganismsbeing investigated are grown for about 24 hours in a suitable medium(e.g. heart infusion broth with glucose, yeast-malt medium) on a shaker(e.g. at 28 or 37 C.). The cells from 10 ml. are obtained bycentrifugation and added to 2 ml. of substrate solution at each ofvarious pHs such as 5 and 7, e.g. to a solution of 500 mcg./ ml.phenoxymethylpenicillin aldehyde (IV) in 20% acetone-80% 0.2 molar pH 5citrate bufier. That mixture of cells and substrate is then returned tothe shaker for a short period of time (e.g. four hours). Aliquots aretaken, diluted (e.g. ten-, fiftyand one hundred-fold) and assayed by atypical penicillin assay, e.g. by the disc method on an agar plateseeded with Penicillin V Aldehyde (G-Phenoxyacetamidopencillanal) (l)6-phenoxacetamidopenicillanic thiol acid, N,N'-dibenzylethylenediamz'ne(DBED) salt.--A mixture of 38.0 g. (0.985 mole) of pencillin V potassiumsalt, 20.6 g. of triethylamine hydrochloride (0.15 mole) and 1400 ml. ofmethylene chloride was stirred for /2 hour at 25. Most of the methylenechloride was then removed under vacuum at 30. Dimethylformamide (DMF),500 ml., was then added and the mixture was cooled to 4. Ethylchloroformate, 9.38 ml. (0.0985 mole), was added and the temperatureimmediately rose to +2.5 The mixture was stirred and cooled down to 5during the next ten minutes forming the mixed anhydride. A solution of 22 g. (0.2 mole) of sodium hydrosulfide (hydrated) in 350 ml. of DMF wasprepared by stirring for ten minutes at room temperature under anitrogen atmosphere. The solution was a turbid yellow, but all chunks ofthe salt had B. subtilis; activity in this assay indicates the presenceof disappeared at this time. The solution of sodium hydro- .9 sulfidewas poured into the mixed anhydride solution, and the resulting darkmixture was stirred at +l to 0 for ten minutes forming the thio acid.The reaction mixture was immediately poured into 3700 ml. of ice water,acidified to pH 2 with ca. 50 ml. of 6 N H 80 and extracted with two1500 ml. portions of ice-cold ether. The combined yellow ether extractswere washed twice with 75 ml. portions of ice water, and then extractedwith two 200 ml. portions of 4.25% NaHCO solution which carried theyellow color into the aqueous layer. The combined basic extracts wereadded to a solution of 36 g. (0.1 mole) of N,N'-dibenzylethylenediaminediacetate in 200 ml. of water forming the bis salt of the thio acid. Thetitle compound crystallized when all of the dissolved ether was removedunder vacuum. It was filtered off, dried briefly on the filter, thenplaced in a 2-liter round-bottom flask. Acetone, 100 mL, was addedfollowed by 200 ml. of ethyl acetate and the mixture was flashed todryness at 33 thereby removing the remaining traces of water. The yellowsolid was partly dissolved in 50 ml. of dry acetone, diluted with oneliter of dry ether and filtered off and dried under vacuum. Yield, 39.5g. Its infrared spectrum showed NH (3350), intense B-lactam (1760),amide (1670 and 1495), -COS (1510) and phenyl absorptions at 1590, 750and 695 cmr (2) 3-(1,3-dibenzyl 2-imitlazolidinyl)-2,2-dimethyl-6-phenoxyacetamidopenam.Commercial Raney active nickel catalyst, No. 28,25 g. (wet weight) was washed three times with 55 ml. portions ofabsolute ethanol, and then with four 55 ml. portions of tetrahydrofuran(THF). The washing procedure is speeded materailly if a bar magnet isused to pull the nickel to the bottom of the breaker after each wash.The nickel was then suspended in 100 ml. of THF and 2.1 ml. (0.035 mole)of glacial acetic acid and 12 g. (0.0050 mole) ofN,N'-dibenzylethylenediamine (DBED) was added and the suspension wasstirred and cooled to A cooled (5) solution of 4.86 g. (0.0100 mole) ofpen V thiol DBED salt in 40 ml. of THF was added and the mixture wasstirred at 2' for hour. The yield and quality of product may possibly besubstantially improved by reducing this reaction time to about 15minutes. The mixture was filtered over diatomaceous earth (Supercel) andthe catalyst was washed with four 30 ml. portions of THF. The THF wasremoved under vacuum at 33 and the dark residue was dissolved in 300 ml.of ether. A considerable amount of gum failed to dissolve. On additionof 50 ml. of water, part of the gum dissolved. In some runs a pink solidseparated at this point and was filtered off. The water layer wasseparated and the ether solution was extracted successively with two 30ml. portions of 5% acetic acid, 50 ml. of water, three 30 ml. portionsof 5% sodium bicarbonate and three 30 ml. portions of Water. The ethersolution was dried briefly over sodium sulfate, treated withdecolorizing carbon and filtered over Supercel which gave a nearlycolorless filtrate. The solvent was flashed ofl at 33, the residue wasflashed with two 100 ml. portions of chloroform to remove moisture andthen thoroughly dried over phosphorus pentoxide at 0.5 mm. The yield ofthe titled compound was 2.23 g. of a hard gum.

(A similar procedure yielded the N,N-diphenylimidazolidinyl analog ofthe title compound which was obtained as a crystalline solid, M.P.174176 sl. dec.)

Analysis.Calcd. for C H N O S: C, 68.15; H, 6.10. Found: C, 67.90; H,6.20.

The infrared spectra of the two imidazolidines were very similar.

(3) 6-pherz0xyacetamidopenicillanuI.-A solution of 2.23 g. (4.00millimole) of the above N,N'-dibenzylimidazolidine in 15 ml. of dryacetone and 45 ml. of ether was treated all at once with a solution of1.67 g. (8.80 millimole) of p-toluenesulfonic acid monohydrate in 7.5ml. of acetone and 22.5 ml. of ether. A heavy pre- 10 cipitate formedinstantly. The suspension was stirred for 15 minutes at 25 and thenfiltered. Addition of 1 ml. of the same toluenesulfonic' acid solutionto the filtrate produced no further precipitate, so the filtrate wasextracted twice with 25 ml. portions of water (the first extract wasstrongly acidic, the second much less acidic), once with 20 ml. of 2%sodium bicarbonate and again with 20 ml. of water. The ethereal solutionwas dried over sodium sulfate, treated with decolorizing carbon, andfiltered over Supercel. Removal of the solvent and through drying undervacuum yielded 1.10 g. of the product, 6-phenoxyacetamidopenicillanal,as a very hard, almost glassy gum. Its infrared spectrum (in methylenechloride) showed a weak OH (3500), NH (3370), intense B-lactam (1780),medium CHO (1730), intense amide (1690 and 1510), phenyl (1595, 1590 and1490) and phenyl-OC (1230). Its NMR spectrum (in CDCl showed all of theexpected sharp resonance lines.

EXAMPLE 3 s CH3 6-(3-phenyl-5-meflzylfsoxazole 4carboxamido)penicillamal.-One hundred grams of wet No. 28 commercialRaney Ni (Raney Co.) was washed three times with ml. portions ofabsolute ethanol, three times with 100 ml. portions of acetone andfinally a fourth portion of 100 ml. of acetone was added and the slurrycooled and stirred at 0 to 5 C. while 7 g. ofN,N-diphenylethylenediamine, 2.4 ml. of glacial acetic acid and asolution of 10 g. of freshly prepared triethylammonium thiol oxacillin[triethylammonium6-(5-methyl-3-phenylisoxazole-4-carboxamido)thiopenicillanate] in 50 ml.of acetone was added in that order. These were added at such a rate asnot to exceed 5 C. The slurry was stirred /2 hour at 2 C., filtered andthe Raney Ni sludge washed with 4 50 ml. portions of acetone. Thecombined filtrates were evaporated to dryness at 25 C. under reducedpressure and the resulting oil dissolved in 500 ml. of dry ether,filtered and the ether washed with 3 X 100 ml. of 5% H PO 3X 100 ml. HO, 3x 100 ml. of 5% NaHCO and 3 X 100 ml. water. The ether solution wasthen dried briefly over Na SO filtered and treated with a concentratedsolution of p-toluenesulfonic acid in acetone until the solution wasstrongly acid to moist pH paper after stirring for 5 minutes. Thecrystalline amine salt was then filtered off and the ether filtratewashed with 3 X 100 ml. water, 3 100 ml. 5% NaHCO and 3X 100 ml. ofwater. The ether solution was dried over Na SO briefly; filtered andstrippel to dryness under high vacuum until the product,6-(3-phenyl-S-methylisoxazole-4-carboxamido)penicillanal, was obtainedas a frothy solid vlgich was scraped out of the flask and found to weighAnalysis.-Calcd. for C I-I N O S: C, 59.25%; H 4.96%; N, 10.9%.

Found: C, 59.70%; H, 5.61%;

Penicillin G aldehyde (6 phenylacetamidopeniciL lanal).-To a precooled,stirred suspension of 13.08 g. (0.03 mole) of penicillinG-triethylammonium salt in 200 ml. of dimethylformamide (DMF) was added3.24 g. (0.03 mole) of ethyl chloroformate dropwise over a ZOminuteperiod. The resulting nearly clear solution was then treated all at oncewith 3 g. of NaSH in 50 ml. of DMF which had been previously dissolvedby pulverizing in a mortar and stirred and cooled in an ice bath until acloudy solution had been obtained. The resulting dark green reactionmixture was stirred one-half hour with the ice bath removed and pouredinto a mixture of 500 ml. benzene, one liter of crushed ice in water and30 ml. of 40% H PO The benzene extract was washed three times with coldwater and partially dried by filtering through sodium sulfate. Thebenzene was then removed under reduced pressure on the flash evaporatorand when the benzene was nearly all removed (avoiding heating) theresulting oil was dissolved in 50 ml. of dry acetone and addedimmediately, dropwise, to a previously prepared stirred suspension of100 g. of No. 28 commercial Raney Ni (Raney Co.), [previously washed 3Xwith absolute ethanol and 4X with dry acetont (200 ml. portions)], 300ml. of acetone, 10.5 g. of N,N'-di-phenylethylenediamine and 3.6 ml. ofglacial acetic acid at 2.0 C. The addition took five minutes and thetemperature rose to 4 C. After stirring an additional A hour the RaneyNi was filtered off through Supercel filter aid and the Raney Ni sludgewas washed 4 with 100 ml. portions of acetone. The combined filtrateswere evaporated to an oil on the flash evaporator and dissolved in oneliter of ether; a precipitate was then filtered off and the ethersolution was extracted 3 with 5% H PO (100 ml.) 3X with Water (100 ml.),3X with 5% NaHCO (100 ml.) and 3 X with water. The ether solution wasdried briefly over Na SO filtered and evaporated under reduced pressureto an oil. The oil was then dissolved in 100 ml. of acetone and 900 ml.of ether added. To this solution was added 6 g. of p-toluenesulfonicacid trihydrate dissolved in 50 ml. of acetone and the resulting slurrystirred for ten minutes, filtered and the filtrate washed three timeswith 200 ml. of water. The ether solution was dried briefly over Na SOfiltered, stripped to dryness and dissolved in 300 ml. of CH Cl andstripped again followed by pumping at high vacuum (0.1 mm. Hg). Theresulting product, penicillin G aldehyde, was thus obtained as a frothysolid which was scraped out, dried over P under vacuum and found toweight 5.6 g.

Alternate procedure.Same as above except the potassium salt ofpenicillin G-thiol acid was used; yield, 1 g.

Analysis.-Calcd. for C H N O S: C, 60.3%; H, 5.70%; N, 8.79%. Found: C,59.8%; H, 6.18%; N, 8.03%.

Alternate p r0cedure.-Same as above except N,N'-dibenzylethylenediaminewas used in place of N,N-diphenylethylenediamine and dilute (2%) aceticacid was used in place of H PO in the acid washes. Tetrahydrofuran wasused as solvent for the reaction and the DBED salt of thethiol-penicillin G was used. The yield was 27 g. (60%) of the crudedibenzylimidazolidine and the aldehyde was liberated as before withp-toluenesulfonic acid. The aldehyde was the same as in previous runs byinfrared spectrum. The yield in this experiment was approximately 50%,an improvement over the methods given above.

EXAMPLE 5 R s CH3 0C 1H-o-NHCHC C CHa H CH. 0=( 3-I I-( 1Ho oPhenethicillin aldehyde (6 or. phenoxypropionamidopenicillanal oror-phen0bcyethylpenicillin aldehyde).-Fifty grams of wet Raney nickelwas washed three times with 100 ml. portions of absolute ethanol andthree times with 100 ml. portions of dry acetone and finally 100 ml. ofdry acetone was added and, while cooling to 0 there was added 4.24 g.(0.02 mole) N,N'-diphenylethylenediamine and 2.4 ml. glacial aceticacid. To this cooled and stirred slurry there was added all at once acooled partial solution of 9.4 g. (0.02 mole of potassiumthio-phenethicillin (potassium6-ot-phenoxypropionamido-thiopenicillanate) in 100 ml. acetone and 2.4ml. glacial acetic acid. The temperature rose to 6 C. and after stirring30 minutes at 0 C. the Raney nickel was removed by filtration and washedfour times with 50 ml. portions of acetone. The combined filtrates wereflash-distilled to dryness under reduced pressure at 20 C. to leave theexpected imidazole as a purple oil which was triturated with 500 ml. dryether and filtered. The ethereal filtrate was washed three times withml. portions of water, three times with 100 ml. portions of 2% NaHCOsolution and three times with water and dried over Na SO To the dryethereal filtrate there was then added a solution of p-toluene sulfonicacid (5 g. in 50 ml. acetone) until the solution was acidic to wet pHpaper. The solid p-toluenesulfonic acid salt ofN,N-diphenylethylenediamine which precipitated was removed by filtrationand the filtrate was treated as before with the acid repeatedly until nomore solid formed upon addition of acid and the ethereal solution wasstill acidic to wet filter paper after standing ten minutes. Theethereal solution containing the product, phenethicillin aldehyde, wasthen washed with 2% NaHCO and water, dried, filtered and the solvent wasremoved by distillation to dryness under high vacuum to leave theproduct as a frothy, amorphous solid weighing 2.9 g. and having aninfrared absorption spectrum consistent with the structure given above.I

Analysis.-Calcd. for C H N O S: C, 58.6; H, 5.80; N, 8.06. Found: C,58.9; H, 6.13; N, 7.52.

Benzyloxypenicillin aldehyde (6 N carbobenzyloxyaminopenicillanaL-To 45g. of previously acetonewashed Raney nickel suspended in 100 ml. ofacetone with 3.7 g. (0.0174 mole) N,N'-diphenylethylenediamine and 2.1m1. of glacial acetic acid, was added 7 g. (0.0174 mole) ofbenzyloxypenicillin thiol (potassium salt) dissolved in 25 ml. ofacetone and 3 ml. of glacial acetic acid. The mixture was stirred forone hour in an icebath and the mixture was filtered. The filtrate wasevaporated to an oil under reduced pressure at 30 and residue wassl-urried with 100 ml. of ether. The ether was washed twice with dilutesodium bicarbonate and several times with water. The ether was driedover anhydrous magnesium sulfate. The drying agent was separated and theether solution treated with a solution of p-toluenesulfonic acid inacetone until there was no more turbidity. TheN,N'-diphenylethylenediamine toluenesulfonate was collected and thefiltrate was washed with water and dried over anhydrous MgSO The solventwas evaporated under reduced pressure to yield 1.5 g. of neutralbenzyloxypenicillin aldehyde.

EXAMPLE 7 6 phthalimidopenicfllin aldehyde (6phthalimidopenicillanal).T0 a solution of 5.0 g. (0.015 mole) of6-phthalimidopenicillanyl alcohol in 50 ml. of anhydrousdimethylsulfoxide was added 10.0 g. (0.049 mole) ofdicyclohexylcarbodiimide and 1.5 g. (0.0075 mole) of pyridiniumtrifiuoracetate. The mixture was swirled in an Erlenmeyer flask for afew seconds, placed in a desiccator under a nitrogen atmosphere, allowedto stand at 25 C. for 17 hours and then filtered and the filtrate throwninto 500 ml. of 50:50 chloroform-water and shaken. The chloroform layerwas separated, washed with water and dried over MgSO for two hours. Thechloroform solution was then filtered and evaporated to EXAMPLE 8 6-(5methyl 3 0-chloropherzylis0xaz0le-4-carb0xamido)penicillanal[S-methyl-3-0-chl0r0phenyl 4 isoxazolylpenicillin rzIzlehyde.Twenty-fiveg. (wet weight) of Raney nickel, No. 28 was washed three times with 55ml. portions of 100% ethanol, followed by four washings with 55 ml.portions of tetrahydrofuran (THF). The nickel was then suspended in 100ml. THF and cooled to 4 and to this there was added 2.1 ml. glacialacetic acid. Potassium6-(5-methyl-3-o-chlorophenylisoxazole-4-carboxamido)thiolpenicillanate(4.5 g.) was dissolved completely in 40 m1. THF and added to the Raneynickel. The reaction mixture was stirred below 0 for 15 minutes. TheRaney nickel was then filtered through diatomaceous earth and washedfive times with 60 ml.

portions of THF. The filtrate was evaporated to dryness.

The resulting oil was partially dissolved in 350 ml. ether. A pink gumappeared which was carried along with the ether. The ether was thenextracted three times with 60 m1. portions of 5% NaHCO and three timeswith 40 ml. portions of water. The ether layer was then separated fromthe gum and dried over magnesium sulfate and charcoal. After filtrationthe ether was evaporated to dryness. The resulting oil could besolidified by dissolving it in methylene chloride or ether andprecipitating with n-pentane. The product,6-(5-methyl-3-o-chlorophenylisoxazole-4-carboxamido)penicillanal [5methyl- 3-o-chlorophenyl-4-isoxazolylpenicillin aldehyde], precipitatedas an amorphous solid, yield 150 mgm. Both IR and NMR spectra showed allof the expected absorptions.

EXAMPLE 9 In the procedure of Example 5 the potassium thiophenethicillinis replaced by an equimolar amount of the potassium salt of the acids6-[a-(2-ch1orophenoxy)propionamido] thiopenicillanic acid,6-[a-(4sultamylphenoxy)-n-butyramido] thiopenicillanic acid,

6- [a-(3,4-dimethoxyphenoxy) -n-pentanoamido] thiopenicillanic acid,

6-[11-(3 methylphenoxy isovaleramido] thiopenicillanic acid,

6-[ot-( l-methylthiophenoxy)propionamido] thiopenicillanic acid,

6-[u-4dirnethylaminophenoxy)-nhexanoamido] thiopenicillanic acid,

6-[a-(2-methoxyphenoxy)-ndecanoarnido] thiopenicillanic acid,

6- rz- 2,4dichlorophenoxy phenylacetamido] thiopenicillanic acid,

6-[a-(2-nitrophenoxy)-fi-phenylpropionamido] thiopenicillanic acid,

6-[a-(2-acetamidophenoxy)-'y-phenylbutyramido] thiopenicillanic acid,

6 OL- 2,4-dimethylphenoxy) -n-butyramido] thiopenicillanic acid,

6 [0L- 4-isopropylphenoxy propionamido] thiopenicillanic acid,

6- a- 3 -bromophenoxy) -n-butyramido] thiopenicillanic acid,

6-[u- (2-iodophenoxy)phenylacetamido] thiopenicillanic acid,

6-[a-(Z-diethylaminophenoxy)isovaleramido] thiopenicillanic acid,

6[w(3,5-dichlorophenoxy)isohexanoamid-o] thiopenicillanic acid,

6- oz- 4-cyclohexylphenoxy propionamido] thiopenicillanic acid,

6-[ot-phenoxy-isovaleramido] thiopenicillanic acid,

6-[aphenoxy-n-decanoamido] thiopenicillanic acid,

6 [or-phenoxy-v-phenylbutyramido] thiopenicillanic acid,

6- [a- 2-benzylphenoxy) -n-butyramido] thiopenicillanic acid,

6[a-(Z-trifluorornethylphenoxy)propionamido] thiopenicillanic acid, and

6- ot-(4-fluorophenoxy) propionamido] thiopenicillanic acid,

respectively, to produce 6- Ot- 2-chlorophenoxy propionamido]penicillanal,

6- cc- (4-sulfamylphenoxy) -n-butyramid-o penicillanal,

6-[a-(3,4-dirnethoxyphenoxy) -n-pentanoamido] penicillanal,

6- w 3 -methylphenoxy) isovaleramido] penicillanal,

6- [w (4-methylthiophenoxy propionamido] penicillanal,

6- oc- 4-dimethylaminophenoxy) -n-hexano amido] penicillanal,

6- [a- Z-methoxyphenoxy) -n-decanoamido] penicillanal,

6-[a-(2,4-dichlorophenoxy)phenylacetamido] penicillanal,

6- u- Z-nitrophenoxy) -,B-phenylpropionamido] penicillanal,

6- OL- 2-acetamidophenoxy) -'y-phenylbutyramido] penicillanal,

6- 0L- 2,4-dimethylphenoxy) -n-butyran1i-do] penicillanal,

6- a-(4-isopropylphenoxy)propionamido] penicillanal,

6-[a-(3-bromophenoxy)-n-butyramido] penicillanal,

6- a- 2-iodophenoxy phenylacetamido] penicillanal,

6-[a-(Z-diethylaminophenoxy)isovaleramido] penicillanal,

6- [oc- 3,5 -dichlorophenoxy isohexano amido] penicillanal,

6- 0!.- (4-cyclohexylphenoxy) propionamido] penicillanal,

6-[u-phenoxy-isovaleramido] penicillanal,

6-[m-phenoxy-n-decanoamido] penicillanal,

6- [or-phenoxy-'y-phenylbutyramido] penicillanal,

6- a- Z-b enzylphenoxy) -n-hutyramido] penicillanal,

6- OL- Z-trifluoromethylphenoxy) propion amido] penicillanal, and

6-[a-(4-fiuorophenoxy)propionamido] penicillanal,

respectively, each of which is isolated as a solid which is converted toan active antibacterial agent upon exposure to an oxidative enzymesystem.

1 EXAMPLE In the procedure of Example 5 the potassium thio-Jhenethicillin is replaced by an equimolar amount of the aotassium saltof the acids,

5- ot-phenylthiopropionamido thiopenicill anic acid,

5- a-paranitrophenylthiopropionamide thiopenicillanic acid,

5- u-parachlorophenylthiopropionamido thiopenicillanic acid,

5- a-phenylthiobutyramido thiopenicillanic acid,

6- a-phenylthiocapro amide thiopenicillanic acid,

6- a-phenylthioisovaleramido thiopenicillanic acid,

6- a- 4-t-butylphenylthio propionamido] thiopenicillanic acid,

6-[a-ortho-tolylthiopropionamido]thiopenicillanic acid,

6 a-ortho-nitrophenylthiopropionamido thiopenicillanic acid,

6- u-parachlorophenylthiobutyramido thiopenicillanic acid,

6- oc- 3 ,4,5-trichlorophenylthio propionamide] thiopenicillanic acid,

6- a- 3 -trifluoromethylphenylthio butyramido] thiopenicillanic acid,

6- a-p arabromophenylthioisovaleramido) thiopenicillanic acid,

6- a-paraphenylphenylthiopropionamido) thiopenicillanic acid,

6- a- 4-methoxypheny1thio) caproamido 1 thiopenicillanic acid,

6- OL- 4-cyclohexylphenylthio butyramido] thiopenicillanic acid,

6- a-phenylthio-a-cyclohexylacetamido thiopenicillanic acid,

6- a-phenylthio-a-cyclopentylacetamide thiopenicillanic acid,

6- a- 2,4-dichlorophenylthio capro amide] thiopenicillanic acid,

6- oc- 2,4-diisoamylphenylthio propionamido] thiopenicillanic acid 6- w4-benzy1phenylthio propionamido 1 thiopenicillanic acid,

6- [u-(4-su1famy1phenylthio )butyramido] thiopenicillanic acid,

6- [ot-(2-al1yloxyphenylthi0 )propionamido] thiopenicillanic acid,

6- oc- 4-ally1phenylthio isovaleramido 1 thiopenicillanic acid,

6- u- (4-dimethylaminophenylthio propionamido] thiopenicillanic acid,

6- a- 2,5 -dichloropheny1thio butyramido] thiopenicillanic acid,

6- [a- 2-iodophenylthio propionamido 1 thiopenicillanic acid,

6- oc- 2-acetamidophenylthio propionamido] thiopenicillanic acid,

6- oc- 4-diethylaminophenylthio propionamido] thiopenicillanic acid, and

6- a- 3-fiuorophenylthio butyramido] thiopenicillanic acid,

respectively, to produce 6-( a-phenylthiopropionamido penicillanal,

'6- a-paranitrophenylthiop ropionarnido pencillanal,

6- ot-parachlorophenylthiopropionamido) penicillanal,

6- oc-PhGllYl'thiOblltYlflIIlidO penicillanal,

6- a-phenylthiocaproamido penicillanal 6- a-phenylthioisovaleramidopenicillanal,

6- u- 4-t-butylphenylthio propionamido 1 penicillanal,

6- u-ortho-tolylthiopropionamido 1 penicillanal,

6- a-ortho-nitrophenylthiopropionamido penicillan a1,

6- a-parach10rophenylthiobutyramido penicillanal,

6- oc- 3 ,4,5 -trich1orophenylthio propionamido] penicillanal,

6- oc- 3 -trifiuoromethylphenylthio butyramido] penicillanal,

6- u-parabromophenylthioisovaleramido penicillanal,

6- a-paraphenylphenylthiopropionamido penicillanal,

6- oc- 4-methoxyphenylthio caproarnido 1 penicillanal,

6- a-phenylthio-u-cyclohexylacetamido penicillanal,

6- a-phenylthio-u-cyclopentylacetamido penicill anal,

6- zx- (2,4-dichloropheny1thio caproamido1penicillanal,

6- ot-(2,4-disoamylphenylthio propionamido] penicillanal,

6- a-(4-benzylphenylthio propionamido1penicillanal,

6- a- 4-sulfamylphenylthio butyramido 1 penicillanal,

6- [ot-(2-ally1oxyphenylthio)propionamido]penicillanal,

6- [a- (4-allylpheny1thio isovaleramido1penicillanal,

6- a- 4-dimethylaminophenylthio propionamido] penicillanal,

6- [oc- 2,5 -dichlorophenylthio butyramido 1 penicillanal,

6- oc- 2-iodophenylthio propionamido penicillanal,

6- a- Z-acetamidophenylthio propionamido 1 penicillanal,

6- [a-( 4-diethylaminophenylthio) propionamido] penicillanal, and

6- [ca-( 3-fluorophenylthio butyramido1penicillana1,

respectively, each of which is is isolated as a solid which is convertedto an active antibacterial agent upon exposure to an oxidative enzymesystem.

EXAMPLE 1 1 In the procedure of Example 5 the potassiumthiophenethicillin is replaced by an equimolar amount of the potassiumsalt of the acids,

D,L-6- [ot-amino- 3 -thienyl acetamido] thiopenicillanic acid,

6- a-amino- 5 -ethyl-2-thieny1 acetamido 1 thiopenicillanic acid,

6- [0L-aI'1'lil'10-( S-methyl-Z-thienyl) acetamido] thiopenicillanicacid,

6- a-amino- 5 -t-butyl-2-thienyl) acetamido] thiopenicillanic acid,

6- [a-amino- 2,5 -dimethyl-3-thienyl) acetamido] thiopenicillanic acid,

6-[a-amino- (5 -chloro-2-thienyl) acetamido] thiopenicillanic acid,

6- a-amino- (5 -bromo-2-thienyl) acetamido] thiopenicillanic acid,

6- a-amino- 5 -phenyl-3 -chloro-2-thienyl) acetamido] thiopenicillanicacid,

6- [cc-amino 3 ,5 -dimethyl-2-thienyl acetamido] thiopenicilianic acid6- a-amino 5 -cyclohexyl-2-tl1ienyl acetamido] thiopenicillanic acid,

6- ot-amino- 5 -diethylamino2-thienyl) acetamido] thiopenicillanic acid,

6- a-amino- 4-methylsulfony1-2-thienyl) acetamido] thiopenicillanicacid,

6- [a-amino- 3 -ethylthi0-2-thienyl acetamido] thiopenicillanic acid,and

6 [a amino (4 cycloheptyloxy 2 thienyl)acetamido]thiopenicillanic acid,

respectively, to produce 23 (N,N-tetramethylene-N'-phthalamidopenicillanal, {N-(Z-phenylethyl) -N'-phthalarnido1penicillanal,(N,N-pentamethylene-N'-phthalamido penicillanal,-(N-isopropyl-N-phthalamido penicillanal,(N-u-methylbenzyl-N-phthalamido penicillanal,-(N-hexamethylene-N'-phthala'mido) penicillanal, -(N-tetrahydrofurfuryl-N'-phthalamido penicillanal,-(N-morpholino-N'-phthalamido penicillanal,

- [N (2,6 dimethylmor pholino) -N'-phthalamido penici'llanal,(N,N-di-n-butylphthalamido penicillanal, [o (2-methyl-1,2,3,4-tetrahydroquinolyl-carb onyl) benzamido1penicillanal,(N,N-diallyl-N-phtha1amid0 penicillanal, (N,N-diisopropyl-N-phthalamidopenicillanal, (N,N-diethyl-N-phthalamido penicillanal,(N,N-dimethyl-N'-phthalamido penicillanal, (N-dodecyl-N-phthalamidopenicillanal, (N-t-butyl-N'qahthalamido penicillanal,-N-methyl-N-phthalamido penicillanal, (N-ethyl-N'-phthalamidopenicillanal, N-isobutylphthalamido penicillanal,(N-a-methylpropylphthalamido penicillanal,N-a-methylphenethyLN'-phthalaimido penicillanal,N-2-methoxyphenyl-N'-phthaiamido penicillanal,(N-phenyl-N-ethyl-N-phtha1a1nido penicillanal, [N- Z-methyl-S-ethy1piperidino) -N --phthalarnido] penicillanal, N-pyrrolidino-N-phthalamido penicillanal,-[N-(2-methylpyrrolidino)-N-phthalamido1penicillanal, [N- 2,5-dimethylpyrr-olidino) -N'-phthalamido1 penicillanal,(N,N-heXamethylene-N'-phthalamido penicillanal, i-[N-( 1,1,3,3-tetrarnethylbutyl) -N-phthalamido] penicillanal, i-(N-cyclohexyl-N'-phthalamido penicillanal, i- [N- (3 -morpholinopropyl)-N-phthalamido1penicillanal,

and i- [N-(Z-methylpiperidino -N'-phthalamido1penicillanal,

espectively, each of which is isolated as a solid which s converted toan active antibacterial agent upon exposure an oxidative enzyme system.

EXAMPLE 16 In the procedure of Example 5, the potassiumthioahenethicillin is replaced by an equimolar amount of the potassiumsalt of the acids,

- oc- 2-chlorophenoxy) acetamido 1 thiopenicillanic acid,

3-[ot-(4-SU1-f2lII1Y1Ph6IIOXY) acetamido] thiopenicillanic acid,

5- a- 3 ,4-dimethoxyphenoxy) acetamido 1 thiopenicillanic acid 5- a- 3-methylphenoxy) acetamido 1 thiopenicillanic acid,

5- oc- (4-rnethylthiophenoxy) a-cetamido thiopenicillanic acid,

5- u-4-dimethylaminophenoxy) acetamido 1 thiopenicillanic acid.

5- a-(2-methoxyphenoxy) acetamido] thiopenicillanic acid,

5- [a- 2,4-dichlorophenoxy) phenoxyacetamido] thiopenicillanic acid,

6- oc- 2-nitrophenoxy) acetamido] thiopenicillanic acid,

6- a- Z-acetamidophenoxy) acetamido] thiopenicillanic acid,

6- u- (2,4-dimethylphenoxy) acetamido] thiopenioillanic acid,

6- a- (4-isopropylphenoxy) acetamido thiopenicillanic acid,

6- oc- 3-bromophenoxy) acetamido1thiopenicillanic acid,

6- oc- (2-iodophenoxy phenoxyacetamido 1 thiopeniciilanic acid,

6- oc- Z-diethylaminophenoxy) acetamido 1 thiopenicillanic acid,

6- a- 3 ,5 -dichlorophenoxy) acetamido] thiopenicillanic acid,

6- 0C- 4-cyclohexylphenoxy) acetamido 1 thiopenicillanic acid,

6- a-phenoxyacetamido thiopenicillanic acid,

6- [a-(Z-benzylphenoXy) acetamido] thiopenicillanic acid,

6- oz- (Z-trifiuoromethylphenoxy) acetamido 1 thiopenicillanic acid,

6- [a-(4chlorophenoxy) acetamido] thiopenicillanic acid,

6-[a-(4-nitrophenoxy) aceta mido1thiopenicillanic acid,

6- 1 a- (4-bromophenoxy) acetamido]thiopenicillanic acid,

6- [a-(4-t-butylphenoxy) acetamido] thiopenicillanic acid,

6- [oc- (4-trifiuoromethylphenoxy) acetamido1thiopenicillanic acid,

6-[ot-(3-fluorophenoxy) acetamido]thiopenicillanic acid,

6- u- (4-sulfamylphenoxy) acetamido] thiopenioillanic acid,

6- [oc- 2-benzylphenoxy) acetamido 1 thiopeni'cillanic acid,

6- oc- 3 -methoxyphenoxy) acetamido] thiopenicillanic acid,

6-[a-(2-iodophenoxy)acetamido]thiopenicillanic acid,

6- oc- (3 -diethylaminophenoxy) acetamido 1 thiopenicillanic acid, and

6- oc- (2,4-diisoamylphenoxy) acetamido] thiopenicillanic acid,

respectively, to produce 6- a- 2-chlorophenoxy) acetamido1penicillanal,

6- [a-(4-sulfarnylphenoxy) acetamido1penicillan'al,

6- [oz-( 3 ,4-dimethoxyphenoxy) acetamido1penicillanal,

6- a- 3 -methylphenoxy) acetamido1penicillanal,

6[a-(4-methylthiophenoxy) acetamido1penicillanal,

6- oc- (4-dimethylaminophenoxy) acetamido 1 penicillanal,

6- [w (Z-methoxyphenoxy) acetamido1penicillanal,

6- a- 2,4-dichlorophenoxy) phenoxyacetarnido] penicillanal,

6- a- 2-nitrophenoxy) acetamido] penicillanal,

6- :x- 2-acetamidophenoxy) aceta mido1penicillanal,

6- cc- 2,4-dirnethylphenoxy acetamido 1 penicillanal,

6- [oc- (4-isopropylphenoxy) acetamido1penicillanal,

6- OC- 3 -bromophenoxy) acetamido1penicillanal,

6- a- 2-iodophenoxy) phenoxyacetamido] penicillanal,

6- [a- (Z-diethylaminophenoxy) acetamido1penicillanal,

6- a- 3,5 -dichlorophenoxy) acetamido 1 penicillanal,

6- oc- 4-cyclohexylphenoxy acetamido1penicillanal,

6- a-phenoxyacetamido penicillanal,

6- oc- 2-benzylphen0xy acetamido] penicillanal,

6- ov- (Z-trifluoromethylphenoxy acetamido] penicillanal,

6- oc- (4-chlorophenoxy) acetamido] penicill anal,

6- [a- (4-nitrophenoxy) acetamid01penicillanal,

6- [ot-(4-bromophenoxy acetamido1penicillanal,

6- [a-(4-t-butylphenoxy)acetamido1penicillanal,

6- [a-(4-trifiuor0methylphenoxy) acetamido1penicillanal,

6- oc- 3 -fluorophenoxy) acetamido1penicillanal,

6- [oc- (4-sulfamylphenoxy) acetamid01penicillanal,

6- [or-(l-benzylphenoxy) acetamido1penicillanal,

6- [a-(3-methoxyphenoxy) acetamido1penicillanal,

6- [a- (2-i0dophenoxy) acetamido1penicillanal,

6- a- 3 -diethylaminophenoxy) acetamido1penicillanal,

and

6- [a-(2,4-diisoamylphenoxy) acetamido1penicillanal,

respectively, each of which is isolated as a solid which is converted toan active antibacterial agent upon exposure to an oxi-dative enzymesystem.

EXAMPLE 17 In the procedure of Example 5, the potassiumthiophenethicillin is replaced by an equimolar amount of the potassiumsalt of the acids,

6- a-phenylthioacetamido thiopenicillanic acid,

6- oc-p aranitrophenylthioacetamido thiopenicillanic acid,

6- a-parachlorophenylthioacetamido )thiopenicillanic acid,

6- oc- (4-tertiary-butylphenylthio) acetamido] thiopenicillanic acid,

6- a-ortho-tolylthioacetamido thiopenicillanic acid,

6- a-ortho-nitrophenylthioacetamido) thiopenicillanic acid,

6- a-( 3,4,5 -trichlorophenylthio acetamido] thiopenicillanic acid,

6- a- (3 -trifiuoromethylphenylthio acetamido] thiopenicillanic acid,

6- a-parabromophenylthioacetamido thiopenicillanic acid,

6- a-paraphenylphenylthioacetamido thiopenicillanic acid,

6- [a- (4-methoxyphenylthio acetamido]thiopenicillanic acid,

6- a- (4-cyclohexylpheny1thio acetamido] thiopenicillanic acid,

6- [a-2,4-dichlorophenylthio acetamido thiopenicillanic acid,

6- [oe-(2,4-diisamylphenylthi0) acetamido1thiopenicillanic acid,

6- [a- 4-benzylphenylthio) acetamido] thiopenicillanic acid,

6- [a-(4-sulfamylphenylthio) acetamido] thiopenicillanic acid,

6- [a- (2allyloxyphenylthio) acetamido] thiopenicillanic acid,

6'[a(4-a1lylphenylthio acetamido]thiopenicillanic acid,

6- [u- (4-dimethylaminophenylthio) acetamido1thiopenicillanic acid,

6- [ix- 2,5 -dichloropheny1thio acetamido] thiopenicillanic acid,

6- [a-(Z-iodophenylthio) acetamido1thiopenicillanic acid,

6- [a- (Z-acetamidophenylthio) acetamido] thiopenicillanic acid,

6- a- (4-diethylaminophenylthio acetamido] thiopenicillanic acid, and

6- [cz- (3-fluorophenylthio) acetamido] thiopenicillanic acid,

respectively, to produce 6- u-phenylthioacetamido penicillanal,

6- a-paranitrophenylthioacetamido penicillanal,

6-( a-parachl0rophenylthioacetarnido)penicillan a1,

6- [oc- (4-tertiary-butylphenylthio) acetamido1penicillanal,

6- a-ortho-tolylthioacetamido penicillanal,

6- wortho-nitrophenylthioacetamido) penicillanal,

6-[11- (3 ,4,5 -trichlorophenylthio) acetarnido] penicill anal,

6- [a-(3-trifluoromethylphenylthio) acetamido1penicillanal,

6-( a-parabromophenylthioacetamido penicillanal,

6- a-paraphenylphenylthioacetamido)penicillanal,

6- [oc- (4-methoxyphenylthio) acetamido1penicillanal,

6- [a- (4-cyclohexylpheny1thio) acetamido1penicillanal,

6- [a- 2,4-dichloropheuylthio acetamido] penicillanal,

6- [oc- (2,4-diisoamy1phenylthio acetamido penicillanal,

6- [a- (4-1bSHZY1PhEI1Y1thiO) acetamidoJpenicillanal,

6- [oc- 4-sulfarnylphenylthio) acetamido] penicillanal,

6- a- 2-allyloxyphenylthio) acetamido] penicillanal,

6-[a-(4-al1ylphenylthio) acetamido1penicillanal,

6- oc- 4-dimethylaminophenylthio acetarnido] penicillanal,

6- [oc- 2,5 -dichlorophenylthio acetamido] penicillanal,

6- [oc- (2-iodophenylthio) acetamido1penicillanal,

6- [0t- Z-acetamidophenylthio acetamido] penicillanal,

6- [a- (4-diethylaminophenylthio) acetamido1penicillanal,

and

6- [w 3-fluorophenylthio acetamido] penicillanal,

respectively, each of which is isolated as a solid which is converted toan active antibacterial agent upon eX- posure to an oxidative enzymesystem.

EXAMPLE 18 6-[a-(4-nitrophenyl) acetamido]thiopenicillanic acid, 6- cc-(4-bromopheny1 acetamido] thiopenicillanic acid, 6- [u-(4-t-butylphenyl)acetamido1thiopenicillanic acid,

26 6- [a- (4-trifiuoromethylphenyl) acetamido1thiopenicillanic acid, 6-[w 3-fluorophenyl) acetamido] thiopenicillanic acid,6-[u-(4-sulfamylphenyl) acetamido]thiopenicillanic acid, 6- [a-(2-benzylphenyl) acetamido] thinopenicillanic acid,6-[a-(3-methoxypheny1) acetamido]thiopenicillanic acid,6-[a-(2-iodophenyl) acetamido] thiopenicillanic acid, 6- [oc-S-diethylaminophenyl) acetamido] thiopenicillanic acid, and 6-[a-(2,4-diisoamylpheny1) acetamido]thiopenici1lainc acid,

respectively, to produce 6- a- (4-nitrophenyl) acetamido]penici11anal,

6- [a-(4-bromophenyl) acetamido1penicillana1,

6- 0L- (4-t-butylphenyl) acetamido] penicillanal,

6- 0t- 4-trifluoromethylphenyl) acetamido penicillan a1, 6-[ac-(3-flll0l0pl16I1Y1) acetamido1penici1lanal,

6- oc- 4-sulfamy1pheny1) acetamido] penicillanal,

6- a- (Z-benzylp-henyl) acetamido] penicillanal,

6- a- 3-methoxyphenyl) acetamido] penicillanal,

6- [a- 2-i0 dophenyl acetamido] penicillanal,

6- [o.-(3-diethylaminopheny1) acetamido1penicillanal, and 6- [oc-(2,4-diisoamylphenyl acetamido1pencillanal,

respectively, each of which is isolated as a solid which is converted toan active antibacterial agent upon exposure to an oxidative enzymesystem.

EXAMPLE 19 In the procedure of Example 5, the potassiumthiophenethicillin is replaced by an equimolar amount of the potassiumsalt of the acids, i

6-( a-phenylacetamido thio-penicillanic acid,

6- a-paranitrophenylacetamido t'hiopenicillanic acid,

6- a-parachlorophenylacetamido thiopenicillanic acid,

6- oz- 4-tertiary-butylphenyl) acetamido] thiopenicillanic acid,

6-( a-ortho-tolylacetamido thiopenicillanic acid,

6- u-ortho-nitrophenylacetamido) thiopenicillanic acid,

6- a- 3,4,5 -trichloropheny1) acetamido] thiopenicillanic acid,

6- a- 3-trifluoromethylphenyl) acetamido] thiopenicillanic acid,

6- a-parabromophenylacetamido) thiopenicillanic acid,

6- (a-paraphenylphenylacetamido thiopenicillanic acid,

6- a- (4-methoxyphenyl) acetamido] thiopenicillanic acid,

6- oc- (4-cyclohexy1phenyl) acetamido] thiopenicillanic acid, I

6- a,- (2,4-dichloropheny1) acetamido] thiopenicillauic acid,

6- [oc-( 2,4-diisoamylphenyl) acetamido] thiopenicillanic acid,

6- [oc- (4-sulfamy1pheny1 acetamido] thiopenicillanic acid,

6-[ot-(2 -allyloxyphenyl) acetamido]thiopenicillanic acid,

6- a-(4-allyl-phenyl) acetamido] thiopenicillanic acid,

6- [a-(4-dimethylaminophenyl) acetamido1thiopenicillanic acid,

6- [a- (2,5dich10ropheny1) acetamido] thiopenicillanic acid,

6- oc- (2-iodopheny1 (acetarnido thiopenicillanic acid,

6- [a-(2-acetamidophenyl) acetamido] thiopenicillanic acid,

6- O6- 4-diethylaminop-henyl) acetamido] thio-penicillanic acid, and

6-[a-(3-fluorophenyl) acetamido]thiopenicillanic acid,

respectively, to produce 6- a-phenylacetamido )penicillanal,

6-( aparanitrophenylacetamido )penicillanal,

6- oc-p arachlorophenylacetamido penicillanal,

6- [a-(4-tertiary-buty1phenyl) acetamido1penicillanal, 6-a-ortho-tolylacetamido penicillanal,

6- (a-ortho-nitrophenylacetamido) penicillanal,

3-isopropyl-5-phenyl-4-isoxazolylpenicillin aldehyde, 3-tert.butyl--methyl-4-is-oxazolylpenicillin aldehyde,3-methyl-S-p-trifluorornethylphenyla4-isoxazolylpenicillin aldehyde,

3-methyl-5-cyclohexyl-4-isoxazolylpenicillin aldehyde,3-cyclohexyl-5-methyl-4-isoxazolylpenicillin aldehyde,3-a-furyl-5-methyl-4-isoxazolylpenicillin aldehyde, and3-a-thienyl-5-methyl-4-isoxazolylpenicillin aldehyde,

respectively, each of which is isolated as a solid which is converted toan active antibacterial agent upon exposure to an oxidative enzymesystem;

EXAMPLE 21 In the procedure of Example 5, the potassiumthiophenethicillin is replaced by an equimolar amount of the potassiumsalt of the acids,

respectively, to produce,

3,5-diphenyl-4-isoxazolylpenicillin aldehyde,3-methyl-5-pheny1-4-isoxazolylpenicillin aldehyde,3,5-dimethyl-4-isoxazolylpenioillin aldehyde,5-benzyl-3-methyl4-isoxazolylpenicillin aldehyde,3-methyl-S-styryl-4-isoxazolylpenicillin aldehyde, S-tert.butyl-3-phenyl-4-isoxazolylpenicillin aldehyde,5-(2-furyl)-3-methyl-4-isoxazolylpenicillin aldehyde,3-methyl-5-(3',5'-d-imethyl-4'-isoxazolyl)-4-isoxazolylpenicillinaldehyde, B-methyl-S-(Z-thienyl)-4-isoxazolylpenicillin aldehyde,3-(p-chlorophenyl)-5-methyl-4-isoxazolylpenicillin aldehyde, 3-methyl-S-methylmercapto-4-isoxazolylpenicillin hyde,

alde

S-(p-chlorophenyl)-3-methyl-4-isoxazolylpenicillin aldehyde, 3 methylS-(o-nitrophenyl)-4-isoxazolylpenicillin aldehyde,5-isopropyl-3-methyl-4-isoxazolylpenicillin aldehyde and5-methy1-3-(p-chlorophenyl)-4-isoxazolylpenicillin aldehyde,

respectively, each of which is isolated as a solid which is converted toan active antibacterial agent upon exposure to an oxidative enzymesystem.

EXAMPLE 22 (1) 6-phenoxyacelamidopenicillanic thiol acid potassiumsalts.-A solution containing 35.04 g. (0.100 mole) of penicillin V(phenoxymethylpenicillin) 14.02 ml. (0.10 mole) of dry triethylamine,and 508 ml. of pure, dry DMF (dimethylformamide) was stirred and cooledto -3 C. Ethyl chloroforrnate (9.51 ml.; 0.10 mole) was added and theresulting solution was stirred at 0 for 10 minutes. A solution of 22.0g. (0.2 mole) of sodium hydrosulfide (hydrated) in 358 ml. of pure, dryDMF was prepared by stirring at 25 C. under a nitrogen atmosphere for 10minutes. This turbid yellow solution was added to the mixed anhydridesolution all at once and the cooling bath was removed. After stirringfor 10 minutes,

the reaction mixture was poured into 3.0 liters of ice water. Thesolution was acidified to pH 2.0 with 6 N sulfuric acid and extractedwith three one-liter portions of cold ether. The combined ethersolutions were washed twice with 500 ml. portions of ice water, driedvery briefly over sodium sulfate and filtered. Addition of 20.5 ml. ofan by weight solution of potassium-Z-ethylhexanoate (KEH) in dryn-butanol (diluted to 40 ml. with dry ether) precipitated the product,which was collected by filtration, Washed with dry ether and dried,yield 32.7 g. (81%). The product was recrystallized by dissolving in aminimum of cold water (ca. 40 ml.), adding ca. 500 ml. of n-butanol, andremoving the water-butanol azeotrope in vacuo at 33 C. until thesolution was entirely dry. The purified yield was 28.2 g. (70%). Theinfrared absorption spectrum was identical to that of the originalsample of this material for which excellent analytical data had alreadybeen obtained.

(2) 3 (1,3 diphenyl-2-imidaz0lidinyl)-2,2-dimethyl-6-phenoxyacetamidopenam.-A suspension of 52.0 g. of Raney active nickelNo. 28 (washed by decantation three times with ml. portions of absoluteethanol and then four times with 105 ml. portions of THF) in 160 ml. oftetrahydrofuran (TI-1 F) containing 3.20 ml. of glacial acetic acid,5.00 g. (0.0235 mole) of N,N'-diphenylethylenediamine, and 15.0 ml. ofwater was cooled to 0 C. To this vigorously stirred solution was added asolution of 6.35 g. (0.0157 mole) of 6-phenoxyacetamidopenicillanicthiol acid potassium salt in 60 ml. of THF containnig 6.25 ml. of water.An eifervesce was noted, which subsided after about one minute. Themixture was stirred at 60 C. for 15 minutes. The catalyst was thenfiltered oit over diatomaceous earth (Supercel), and the nickel waswashed on the filter with five 50 ml. portions of THF. The solvent wasremoved in vacuo from the pale yellow filtrate, and the residue wasflash-distilled with two 100 ml. portions of dry ethyl acetate at 33 C.to remove traces of moisture. Five hundred ml. of anhydrous ether wasadded and the turbid solution was refluxed for onehalf hour. During thisperiod of reflux, a white, crystalline precipitate formed. At the end ofthe reflux period, the warm solution was filtered, and the solids andfiltrate were worked up separately.

The solids were boiled briefly with 100 ml. of ethyl acetate and the hotmixture was filtered. On thorough cooling, the filtrate yielded 2.70 g.of white crystals, M.P. 174-175 C. Then the filtrate from this portionof the product was freed of solvent in vacuo and the residue was treatedwith 15 ml. of dry ether, a further quantity of the product, 1.42 g.,was obtained as a highly crystalline but slightly tan solid, M.P. l67l70C.

The filtrate was extracted with 20 ml. of 10% phosphoric acid, whichcaused N,'Ndiphenylethylenediarnine phosphate to crystallize. It wasfiltered off, the aqueous phase was separated, and the other solutionwas again extracted with 20 ml. of 10 phosphoric acid and washed withthree 100 ml. portions of water. It was dried over sodium sulfate,filtered, flashed dry and flashed with ethyl acetate. The addition of 10ml. of dry ether to the small residue caused 0.25 g. of product tocrystallize, M.P. 164168 C.

The total yield was 4.37 g. (52.5%) of the title compound.

(3) 6 phenoxyacetamidopenicillanaL-A solution of 10.5 g. of pure3(1,3-diphenyl-2-imiclazolidinyl)-2,2-dimethyl-6-phenoxyacetamidopenam(0.189 mole) was prepared in 98.5 ml. dry acetone, 98.5 .ml. ofmethylene chloride and 298 ml. of anhydrous ether. To this solution wasadded a solution of 7.11 g. (0.374 mole) of ptoluenesulfonic acidmonohydrate in a mixture of 50 ml. of CH Cl 50 ml. of dry acetone, ml.of anhydrous ether, and 1.3 ml. of water. A heavy precipitate formedinstantly, and the slurry was stirred mechanically at 25 for /2 hour.-It was then diluted with 1.1 liter of ether and filtered over Supercel.The clear filtrate was ex- 'acted with two 100 ml. portions of water,dried over soium sulfate, filtered and flashed dry at 33 C. Fifty ml. fdry ether was added and the solution cooled in ice for t least 15minutes. The product crystallized and filtraon yielded 2.08 g. of whiteneedles, M.P. 106108 C. he filtrate was allowed to evaporate with 1.5ml. of H dded at 20 C. for 24 hours. The crystals were filtered nd theyield was 3.90 g. of white needles, M.P. 105104 2. The total yield was5.98 g. (94.5%).

The infrared absorption spectrum was quite sharp, howing NH 3380 c-m.-fl-lactam 1780, aldehyde 1740, mide 1670 and 1525 cmf All of the bandsnoted were -f equal intensity. The aldehyde may be recrystallized cut at33 C. Water (a few ml.) and ordinary ether cetate (three parts per 100ml.) and flashing off the solent at 33 C. Water (a few ml.) and ordinaryether one part per 100 ml.) are added and the resulting soluion isallowed to stand at 20 C. for 24 hours. The prodict will slowlycrystallize.

Analysis.-Calcd for C H N O S- /2H O: C, 55.96; i, 5.57; N, 8.16; S,9.34. Found: C, 55.40, 55.25; H, L63, 5.72; N, 8.08; S, 9.12.

(4) 2,2 dimethyl-3-acetoxymethylene-6-phenoxyacefzmid0penam.A mixture of2.07 g. (0.00604 mole) of )BIllCilllIl V aldehyde hemihydrate, 200 ml.of acetic aniydride, and 3.0 ml. of pyridine was stirred at 25 under 1nitrogen atmosphere for 16 hours. The acetic anrydride and excesspyridine were removed in vacuo at 50 C. The dark residue was dissolvedas far as possible n 150 ml. of carbon tetrachloride and the solutionwas lined with ca. 4 g. of decolorizing charcoal for 45 minites. Thesolution was filtered over Supercel and the iltrate was flashed dry at50 C. In order to codistill ost of the remaining traces of aceticanhydride, 75 ml. of carbon tetrachloride was added and the flashing wasrepeated. The light orange residue was dissolved in a few ml. ofmethylene chloride, flashed to an oil and dried to a friable fluff inhigh vacuum at 25 C. The yield was 1.11 g. (49%) of light orange foam.The infra-red spectrum (5% in CCl shows NH at 3400 cmr ,8- lactam 1800,enol acetate carbonyl 176 2, amide 1700 and 1510 and COC bands at 1230,1190, and 1090 crnf A weak band at 1640 cm.- is ascribed to The NMRspectrum is in full agreement with the enol acetate structure and showsfurther that the product is a mixture of approximately equal amounts ofcis and trans isomers.

Analysis.Calcd for C H N O S: C, 57.43; H, 5.63. Found: C, 57.20; H,5.37.

PREPARATION OF STARTING MATERIALS PENICILLIN THI'OACIDS Another seriesof starting materials used to prepare the compounds of the presentinvention comprises the series of compounds of the formula wherein R isamino, acylarnino, ibenzyloxycarbonylamino, phthalimido or tritylaminoand, particularly, the so-called penicillin thioacids or thiopenicillinsof the formula S CH3 ll in which R represents the side chain of any ofthe known penicillins other than those containing a group which isaltered by reaction with Raney nickel above 0 C. These compounds areusually used in the form of salts, e.g. sodium, potassium.

These compounds exist in tantomeric forms, i.e. the -COSH group may bein the thiol form as or in the thione form as Such thiopenicillins areprepared from penicillins by the methods described below or in US.Patent 2,751,378. In general, to prepare a thiopenicillin, an activeacylating derivative of the starting penicillin such as an anhydride ora mixed anhydride (such as the mixed anhydride with a lower alkyl esterof ethoxy-or isobutoxy-carbonic acid) or an acid chloride is preparedand reacted with a source of sulfhydryl groups, e.g. hydrogen sulfide orsodium hydrosulfide or potassium hydrosulfide.

Additional examples of the preparation of thiopenicillins are asfollows:

Preparation 11.- Preparati0n 0 potassium 6-(DL-uph enoxy pro pionamido)thio p em'cil lanate Dilute sulfuric acid is added to a solution ofpotassium 6-(DL-a-phenoxypropionamido)penicillanate (10 gm., 0.025mole), dissolved in water ml.) until a pH of 2 is attained. Thepenicillin acid is extracted from this solution into ether, washed withwater, and dried over anhydrous magnesium sulfate. The ether isevaporated under reduced pressure to leave a residual oil which isazeotroped three times with ethyl acetate to remove all traces of water.The residue is dissolved in dimethylformamide ml.) and cooled to 5 C. inan ice bath. 2,6-lutidine (2.7 gm., 0.0254 mole) is added, followed bythe dropwise addition of ethyl chloroformate (2.7 gm., 0.0254 mole). Themixture is stirred for 15 minutes and a suspension of sodiumhydrosulfide (5.6 gm., 0.06 mole) in dimethylformamide (100 ml.) isadded all at once. The solution is stirred for one hour, and turns darkbrown. The solution is poured into water (one liter) and acidified to pH2 with dilute sulfuric acid. The 6 (DL-ocphenoxypropionamido)thiopenicillanic acid is extracted into ether,washed with water, and dried over anhydrous magnesium sulfate. Potassium2-ethy1- hexanoate (5 gm.) is added and the crystalline precipitate iscollected and weighs 4.5 gm. Recrystallization from water and acetoneyields 1.1 gm. of the product, potassium 6 (DL aphenoxypropionamido)thiopenicil lanat'e, as colorless plates which arefound to contain the B-lactam ring as shown by infrared analysis, tohave a melting point of greater than 240 C. with decomposition and thefollowing elemental anaylsis:

Calculated for C17H19N2O4S2K'1/2H2OZ C, H, 4.74%; S, 14.9%. Found: C,48.2%; H, 4.52%; S, 13.8%.

Preparation 12 In procedure of Preparation 11, the potassium 6-(u-phenoxypropionamido)pencillanate is replaced by 0.025 mole of thepotassium salt of the acids 6- a- 2-chlorophenoxy propionamido]penicillanic acid,

6 [on (4 sulfamylphenoxy) n butyram-ido]penicil lanic acid,

6 [a (3,4 dimethoxyphenoxy) n pentanoamido] penicillanic acid,

6 [a (3 methylphenoxy)isovaleramido]penicillanic acid,

6 [0: (4 methylthiophenoxy)propionamidoJpen-icillanic acid,

6 d m thylaminophenoxy) n hexanoamido] enicillanic acid,

6 [a (2 inethoxyphenoxy) -1 n decanoamido1penici-llanic acid,

6 [a (2,4 dichlorophenoxy)phenylacetamidoJpenicillanic acid,

6 [a (2 nitrophenoxy) ,8 phenylpropionamido] penicillanic acid,

6 [a (2 a-cetamidophenoxy) 'y phenylbutyramido] penicillanic acid,

6 [a (2,4 dimethylphenoxy) n butyramido1penicillanic acid,

6 [a (4 isopropylphenoxy)propionamido]penicillanic acid,

6 [a (3 bromophenoxy) n butyramido]penicillanic acid,

6 (2 iodophenoxy)phenylacetamido]penicillanic aci 6 [c (2diethylaminophenoxy)isovaleramido]penicillanic acid,

6 [0c (3,5 dichlorophenoxy)isohexanoamido1penicillanic acid,

6 [0c (4 cyclohexyphenoxy)propionamido1penicillanic acid,

6 [a phenoxy isovalerarnido]penicillanic acid,

6-[a-phenoxy-n-deoanoamido] penicillanic acid,

6-[ot-phenoXy-y-phenylb utyramido]penicillanic acid,

5 [a (2 benzylphenoxy) n butyramido]penicillanic acid,

6 [a 4 (Z-trifluoromethylphenoxy)propionamido]penicillanic acid, and

6- a- (4-fluorophenoxy propiona-mido] penicillanic acid,

to produce the potassium salts of 6-[ a-(2-chlorophenoxy) propionaniido]thiopenicillanic acid,

6- [a- (4-sulfamylphenoxy) n-butyramidoJthiopenicillanic acid,

6- [a- 3,4-dimethoxyphenoxy) -n-pentanoamido] thiopenicillanic acid,

6- [a- 3 -met'nylphenoxy isovalerarnido] thiopenicillanic acid,

6-[a-(4-methylthiophenoxy)propionamido] thiopenicillanic acid,

6-[a(4-din1etnylarnmophenoxy) -nhexanoamido] thlopenicillanic acid,

6- ot- (Z-metnoxyphenoxy -n-decanoa-mido] thiopenicillanic acid,

6-[a-(2,4-dichlorophenoxy)phenylacetamido] thiopenicill-anic acid,

6-[tx-(Z-nitr-ophenoxy)p phenylpropionamido] thiopenicillanic acid,

6- a- (Z-acetamidophenoxy) -7-phenylbutyramido] tniopenicillanic acid,

6-[ot-(2,4-dimethylphenoxy)-n-butyramido] thiopenicillanic acid,

6-[ot-(4-isopropylphenoxy)propionarnido1thiopenicillanic acid,

6- a- 3-bromophenoxy) -n-butyramido] thiopenicillanic acid,

6- a- (2-iodophenoxy phenyla-cetamido] thiopenicillanic acid,

6- 0t- (2-diethylaminophenoxy isovaleramido] thiopenicillanic acid,

6-[a-(3,5-dichloro-phenoxy)is-ohexanoamido1 thiopenicillani-c acid,

6- a- (4-cyclohexylphenoxy) propionamido] thiopcnicillanic acid,

6-[a-phenoXy-isovalerarnido1thiopenicillanic acid,

6-[owphenoxy-n-decanoamido1thiopenicillanic acid,

6-[a-phenoxy-v phenylbutyramid-o] thiopenicil'lanic acid,

6- a( Z-benzylphenoxy -n-butyramido] thiopenicillanic acid6-[a-(2-trifiuoromethylphenoxy)propionamido]thiopencillanic acid, and

6-[w (4-fluorophenoxy)propionamido]thiopenicillanic acid,

.34 respectively, each of which is isolated as its solid, waterso'lublepotassium salt.

Preparation 13.-Preparati0n of potassium6-(D-uphenoxypropionamido)thiopcnicillanate Dilute sulfuric acid isadded to a solution of potassium 6-'(D aphenoxypropionamido)penicillanate (5 gm.,

0.0125 mole) in water (150 ml.) until a pH of 2 is attained. Thepenicillin acid is extracted from this solution into ethyl acetate (200ml.), washed with water, and dried over anhydrous magnesium sulfate. Theethyl acetate is evaporated at 35 C. The residue is dissolved indimethylforrnamide (150 ml.) and cooled to 5 C. in an ice bath.2,6-lutidine (1.33 gm., 0.0125 mole) is added, followed by the dropwiseaddition of ethyl chloroformate (1.33 gm., 0.0125 mole). The mixture isstirred for 15 minutes and a suspension of sodium hydrosulfide (2.5 gm.,0.0447 mole) in dimethylformamide :ml.) is added all at once. Thesolution is stirred for 20 minutes and then poured into water (oneliter) and acidified to pH 2 with dilute sulfuric acid. The6-(D-ct-Ph6ll0XY- propionamido)thiopenicillani-c acid that forms isextracted into ether, washed with Water, and dried over anhydrousmagnesium sulfate. Potassium Z-ethylhexanoate (3 gm.) is added and thecrystalline precipitate is collected. Recrystallization from water andn-butanol yields 1.7 gm. of the product, potassium6-(D'a-phenoxypropionamido)thiopenicillan-ate, which is found to containthe fi-lactam ring as shown by infrared analysis, to have a meltingpoint of greater than 195 C. with decomposition and the followingelemental analysis:

Calculated for C H N O S K: C, 48.78%; H, 4.58%; N, 6.7%; S, 15.4%.Found: C, 49.25%; H, 4.68%; N, 6.84%; S, 14.52%.

Preparation 14.---Preparation of potassium6-(L-aphenoxypropionamido)thiopenicillanate Dilute sulfuric acid isadded to a solution of potassium 6(L-u-phenoxypropionamido)penicillanate (5 gm., 0.0125 mole) in waterml.) until a pH of 2 is attained. The penicillin acid is extracted fromthis solution into ethyl acetate (200 ml.), washed with water, and driedover anhydrous magnesium sulfate. The ethyl acetate is evaporated at 35C. The residue is dissolved in dimethylformamide (150 ml.) and cooled to5 C. in an ice bath. 2,6-lutidine (1.33 gm., 0.0125 mole) is added,followed by the dropwlse addition of ethyl chloroformate (1.33 gm.,0.0125 mole). The mixture is stirred for 15 minutes and'a suspension ofsodium hydrosulfide (2.5 gm., 0.0447 mole) in dimethyl'formamide (100ml.) is added all at once. The solution is stirred for 20 minutes andthen poured into water (one liter) and acidified to pH 2 with dilutesulfuric acid. The 6-(L-a-phenoxypropionamido)thiopenicillanic acid isextracted into ether, washed with water, and dried over anhydrousmagnesium sulfate. Potassium 2-ethylhexan0ate (3 gm.) is added and thecrystalline precipitate is collected. Recrystallization from water andn-butan-ol yields 2.5 gm. of the product, potassium6-(L-ot-phenoxypropionamido)thiopenicillanate which is found to have amelting point of greater than 215 C. with decomposition and thefollowing elemental analysis:

Calculated for C17H19N204S2K1 'C, H, N, 6.7%; S, 15.4%. Found: C,50.22%; H, 4.98%; N, 6.77%; S, 14.18%.

Preparation 15.--Preparati0n ofpotassiumo-(aisopropyl-u-phenoxyacetamido)thiopenicillanate Dilutesulfuric acid is added to a solution of potassium6-(a-isopropyl-a-phenoxyacetamido)thiopenicillanate (5.3 gm., 0.0125mole) in water (150 ml.) until a pH of 2 is attained. The penicillinacid is extracted from this solution into ethyl acetate (200 m1.) washedwith water, and dried over anhydrous magnesium sulfate. The ethylacetate is evaporated at 35 C. The residue is disolved indimethylformamide (150 ml.) and cooled to i C. in an ice bath.2,6-lutidine (1.33 gm., 0.0125 mole) s added, followed by the dropwiseaddition of ethyl chlo- 'oformate (1.33 gm., 0.0125 mole). The mixtureis :tirred for 15 minutes and a suspension of sodium hydro- ;ulfide (2.5gm., 0.0447 mole) in dimethylform-amide 1100 ml.) is added all at once.The solution is stirred for 20 minutes and then poured into water (oneliter) and tcidified to pH 2 with dilute sulfuric acid. The6-(asopropyl-a phenoxyacetamido)thiopenicillanic acid is ex- :ractedinto ether, Washed with water, and dried over anhydrous magnesiumsulfate. Potassium Z-ethylhexanoate (3 gm.) is added and the crystallineprecipitate is :ollected. Recrystallization from ethyl acetate andSkellysolve yields 2.3 gm. of the product potassium6-(alsopropyl-a-phenoxyacetamido)thiopenicillanate, which is found tocontain the B-lactam ring as shown 'by infrared analysis and to have amelting point of greater than 170 C. with decomposition.

Preparation 1 6 .--Preparation of potassium 6-[L( -ocphenoxybutyram ido]thio p enici l lanate Potassium 6- [L()-a-phenoxybutyramido]penicillanate (2.08 gm., 0.005 mole) is dissolvedin water (30 ml.) and layered with ethyl acetate. After cooling to 5 C.,dilute phosphoric acid (40%) is added until a pH of 2 is attained. Thepenicillin acid is extracted into the ethyl acetate and a furtherextraction with fresh ethyl acetate is made. The combined extracts arewashed with ice- Water, dried over anhydrous magnesium sulfate, andevaporated to dryness. The residue is dissolved in dimethylformamide (30ml.) and cooled to C. in an ice bath. 2,6-lutidine (0.53 gm., 0.005mole) is added, followed by the dropwise addition of ethyl chloroformate(0.52 gm., 0.005 mole) forming a precipitate. The mixture is stirred forl0'mirrutes at 0 C. and a solution of sodium hydrosulfide trihydrate(1.1 gm., 0.01 mole) in dimethylformamide (30 m1.) is added in oneportion. The resulting green solution is stirred for 25 minutes andpoured into a precooled (10 C.) mixture of water (150 ml.), acidified topH 1.5 with dilute phosphoric acid (40%) and benzene (100 ml.) withvigorous stirring. The 6- [L()-a-phenoxybutyramido]thiopenicillanic acidis extracted into benzene and a further extraction with fresh benzene ismade. The combined extracts are washed and dried. Potassium2-ethylhexanoate (0.005 mole) is added as a 50% solution of potassiumZ-ethylhexanoate in butanol. Skellysolve B (a petroleum ether fractionhaving a boiling point range of from about 60 to about 68 C. consistingessentially of n-hexane) is added to the solution with cooling andshaking until the solution becomes cloudy, and an oil separates. Thesolution is decanted and flashed to dryness. The residue is trituratedwith ether, and a white solid forms which is removed by filtration,washed with dry ether, and dried in vacuo. The product, potassium 6- [L()-a-phenoxybutyramido] thiopenicillanate is found to weigh 2 grams, tohave a melting point of 145 149 C. with decomposition and the structureis confirmed by infrared analysis.

Preparation 17 In the-procedure of Preparation 11, the potassium 6-(a-phenoxypropionarnido)penicillanate is replaced by 0.025 mole of thepotassium salt of 6- a-phenoxy-a-benzylpropionamido penicillanic acid,

6- a-phen'ylthio-a-benzylpropionamido penicillanic acid,

6- a-phenoxy-a-methylpropionamido penicillanic acid,

6- a-phenylthio-a-methylpropionamido penicillanic acid,

6- [a- 2,4-dichl orophenoxy) -a-ethylpropionamido] penicillanic acid,

6- a- Z-chlorophenoxy) -a-methylbutyramido] penicillanic acid,

6 a- (4-nitrophenoxy -a-methyl-n'-butyramido] penicillanic acid,

6- a- 4-b romophenoxy) -a-phenyl-n-valeramido] penicillanic acid,

6-[a-(4-t-butylpl1enoxy) -a-benzyl-n-butyramido] penicillanic acid,

6- [oc- (4-trifluoromethylphenoxy -a-phenyl-n-butyramido] penicillanicacid,

6- a- 3 -fluorophenoxy -a-benzyl-.n-valeramido] penicillanic acid,

6- oc- 4-sulfamylphenoxy) -u-methylpr-opionamido] penicillanic acid,

6- a- Z-benzylphenoxy) -a-methyl-n-butyramido] penicillanic acid,

6 [a- 3-methoxyphenoxy) -a-methyldecanoamido] penicillanic acid,

6- [oc- Z-iodophenoxy) -a-phenylpropionamido] penicillanic acid,

6 [a- B-diethylaminophenoxy) -a-methyl-n-butyramido] penicillanic acid,and

6- rx- 2,4-diisoamylphenoxy) -a-methyl-n-butyramido] penicillanic acid,

to produce the potassium salts of 6- a-phenoxy-a-benzylpropionamidothiopenicillanic acid,

6 a-phenylthio-a-benzylpropionamido) thiopenicillanic acid,

6- a-phenoxy-a-methylp ropionamido thiopenicillanic acid,

6- u-phenylthio-a-methylpropionamido thiopenicillanic acid,

6- oc- 2,4-dichlorophen0xy a-ethylpropion-arnido] thiopenicillanic acid,

6- [a- 2-ch1orop-henoxy) -a-methylbutyramido] thiopenicillanic acid,

6- a- 4-nitrophenoxy -a-methyl-n-butyramido] thiopenicillanic acid,

6- a- (4-bromophenoxy) -a-phenyl-n-valeramido] thiopenicillanic acid,

6- a- 4-t-butylphenoxy) -a-benzyl-n-butyramido] thiopenicillanic acid,

6- [a- 4-trifiuoromethylphenoxy) -a-phenyl-n-butyramido]thiopenicillanic acid,

6- [a- 3-fiuorophenoxy) -a-benzyl-n-valeramido] thiopenicillanic acid,

6-[a-4-sulfarnylphenoxy)-a-methylpropionamido] thiopenicillanic acid,

6- [a- Z-benzylphenoxy -a-methyl-n-butyramido] thiopenicillanic acid,

6- a- 3-rnethoxyphenoxy) -a-methyldecanoamido] thiopenicillanic acid,

6- a- 2-iodophenoxy) -a-phenylpropionamido] thiopenicillanic acid,

6- cx- 3-di-ethylaminophenoxy -a-methyl-n-butyramido] thiopenicillanicacid, and

6-[a-(2,4-diisoamylphenoxy)-a-methyl-n-butyramido] thiopenicillanicacid,

respectively, each of which is isolated as its solid, watersolublepotassium salt.

Preparation 18 In the procedure of Preparation 11, the potassium 6-(a-phenoxypropionamido)penicillanic acid is replaced by 0.025 mole ofthe potassium salt of 6- a-phenylthiopropionamido penicillanic acid, 6-a-p aranitrophenylthiopropionamido penicillanic acid, 6-a-parachlorophenylthiopropionamido penicillanic acid, 6-a-phenylthiobutyrarnido penicillanic acid, 6-( a-phenylthiocaproamidopenicillanic acid, 6- a-phenylthioisovaleramido penicillanic acid, 6- a-(4-t-butylphenylthio propionamido] penicillanic acid, 6-wortho-tolylthiopropionamido penicillanic acid, 6- a-ortho-nitrophenylthiopropionamido) penicillanic acid, 6-a-parachlorophenylthiobutyramido) penicillanic acid,

6-[tx-(3,4,S-trichlorophenylthio)propionarnido] penicillanic acid,

6- oc- 3-trifluoromethylphenylthio) butyramido] penicillanic acid,

6 a-p arabromophenylthioisovaleramido) penicillanic acid,

6- a-paraphenylphenylthiopropionamid'o) penicillanic acid,

6- [a- (4-methoxyphenylthio) caproamido] penicillanic acid,

6- oc- 4-cyclohexylphenylthio) butyramido] penicillanic acid,

6-(a-phenylthio-wcyclohexylacetarnido) penicillanic acid,

6-(a-phenylthio-a-cyclopentylacetamido) penicillanic acid,

6-[a-(2,4-dichlorophenylthio)caproamido] penicillanic acid,

6-[u-(2,4-diisoarny1phenylthio)propionamido] penicillanic acid,

6-[a-(4-benzylphenylthio)propionamido] penicillanic acid,

6-[w(4-sulfarnylphenylthio)butyramido] penicillanic acid,

6-[u-(2-allyloxyphenylthio)propionamido] penicillanic acid,

6-[a-(4-allylphenylthio)isovalerarnido] penicillanic acid,

6-[w(4-dimethylarninophenylthio)propionamido] penicillanic acid,

6-[a-(2,5-di-chlorophenylthio)butyramido] penicillanic acid,

6- a- 2-iodopheny1thio propionamido] penicillanic acid,

6-[a-(Z-acetamidophenylthio)propionamido] penicillanic acid,

6- [t- 4-diethylaminophenylthio propionamido] penicillanic acid, and

6- a- 3-fiuorophenylthio) butyramido] penicillanic acid,

to produce the potassium salts of6-(oephenylthiopropionamido)thiopenicillanic acid,

6-(a-paranitrophenylthiopropionamido) thiopenicillanic acid,

6-( oc-parachlorophenylthiopropionamindo) thiopenicillanic acid,

6-( a-phenylthi obutyramido) tbiopenicillanic acid,

6-(ot-pheny1thiocaproamido)thiopenicillanic acid,

6 (a-phenylthioisovalerami do)thiopenicil1anic acid,

6-[a-(4-t-butylphenylthio)propionamido] thiopenicillanic acid,

6- [a-ortho-tolylthiopropionamido] thiopeni-cillanic acid,

6-(a-ortho-nitrophenylthiopropionarnido) thiopenicillanic acid,

6-( wparachloro-phenylthiobutyramido) thiopenicillanic acid,

6- [a- (3 ,4,5-trichlorophenylthio) propionamido] thiopenicillanic acid,

6-[01-(3 -trifluoromethylphenylthio) butyramido] thiopeni-cillanic acid,

6-(a-parabromophenylthioisovaleramido) thiopenicillanic acid,

6-(a-paraphenylphenylthiopropionarnido) thiopenicillanic acid,

6- [a- (4-methoxyphenylthio cap-roamido] thiopenicillanic acid,

6- O!.- (4-cyclohexylphenylthio butyramido] thiopenicillanic acid,

6 a-phenylthioa-cyclohexylacetamido) thiopenicillanic acid,

6-(oc-phenylthioa-cyc1opentylacetamido) thiopenicillanic acid,

6-[a-(ZA-dichlQrophenyIthio)capro-amido] thiopenicillanic acid,

33 6-[a-(2,4-diisoamylphenylthio)propionamido] thiopenicillanic acid, 6-a- (4-benzylphenylthio propionamido] thiopenicillanic acid,G-[a-(4-sulfalmylphenylthio)butyramido] thiopeni-cillanic acid,6-[a-(2-allyloxyphenylthio)propionarnido] thiopenicillanic acid,6-[oz-(4-allylphenylthio)isovaleramido] thiopeni-cillanic acid,6-[a-(4-dimethylaminophenylthio)propionamido] thiopenicillanic acid,6-[oz-(2,5-dichlorophenylthio)butyramido] thiopenicillanic acid,6-[u-(2-iodophenylthio)propionamido] thiopenicillanic acid,6-[ot-(Z-acetamidophenylthio)propionamido] thiopenicillanic acid,6-[a-(4-diethylarninophenylthio)propi onamido] thiopenicillanic acid,and 6-[a-(3-fluorophenylthio)butyramidoj] thiopenicillanic acid,respectively, each of which is isolated as its solid water,- solublepotassium salt.

Preparation 19 In the procedure of Preparation 16 the 2.08 gm. ofpotassium 6-[L() a phenoxybutyramidojpenicillanic acid is replaced by8.32 grams of the same acid to produce in the first crop 4.0 grams ofthe desired product, potassium 6 [L( ozphenoxybutyramido]thiopenicillanic acid in crystalline form, havingpurity of about (as shown by NMR studies), a specific rotation of 177(C.= /2 H 0) and a melting point range of from 155 to 160 C. withdecomposition.

Preparation 20.Preparati0n of potassium 6- [D -ecplzenoxybulyramido]thiopenicillanale Potassium 6-[D( -0t phenoxybutyramido]penicillanate(3.0 gm.) is dissolved in water (30 ml.) and layered with ethyl acetate.After cooling to 5 C.., dilute phosphoric acid (40%) is added until a pHof 2 is attained. The penicillin acid is extracted into the ethylacetate and a further extraction with fresh ethyl acetate is made. Thecombined extracts are washed with ice-water, dried over anhydrousmagnesium sulfate, and evaporated to dryness. The residue is dissolvedin dimethylformanide (30 ml.) and cooled to 0 C. in an ice bath.2,6-lutidine (0.53 gm., 0.005 mole) is added, followed by the drop Wiseaddition of ethyl chloroforrnate (0.52 gm., 0.005 mole) forming aprecipitate. The mixture is stirred for 10 minutes at 0 C. and asolution of sodium hydrosulfide trihydrate (1.1 gm., 0.01 mole) indimethylformamide (30 ml.) is added in one portion. The resulting greensolution is stirred for 25 minutes and poured into a precooled (5-l0 C.)mixture of water (150 1111.), acidified to pH 1.5 with dilute phosphoricacid (40%) and benzene ml.) with vigorous stirring. The t 6 [D(+) aphenoxybutyrarnido]thiopenicillanic acid is extracted into benzene and afurther extraction with fresh benzene is made. The combined extracts arewashed and dried. Potassium Z-ethylhexanoate (0.005 mole) is added as a50% solution of potassium 2- ethylhexanoate in butanol. Skellysolve B isadded to the solution with cooling and shaking until the solutionbecomes cloudy, and an oil separates. The solution is decanted andflashed to dryness. The residue is triturated with ether, and a whitesolid forms which is removed by filtration, washed with dry ether, driedin vacuo and found to weigh 2.5 gm. The product, potassium 6 [D(+) aphenoxybutyramido]t'hiopenicillanate, is found to have a melting pointof -l45 C. with decomposition, and the structure is confirmed byinfrared analysis.

39 Preparation 21 .-Preparation f potassium 6- [D,L-OL-phenoxypropionamido] thiopenicillanafe Potassium 6[D,L-ot-phenoxypropionamido]penicillante (30.0 gm.) is dissolved in amixture of 100 ml. ice 'ater and 100 ml. trichlorethylene. After coolingthe iixture to C., 6 N hydrochloric acid is added with :irring until apH of 2 is attained. The trichlorethylene base is separated andmaintained at 5 C. The aqueous base is extracted again with 50 ml. oftrichlorethylene and 1e trichlorethylene extracts are combined, mixedwith 40 m. of anhydrous magnesium sulfate and stirred in an ice ath forminutes. The combined extracts are then fil- :red and the filter cake iswashed with 50. ml. of trihlorethylene. The filtrates are placedtogether with 250 31. of dimethylformamide in a one-liter 3-necl edflask vith a drying tube vent and cooled to 3 C. in an icevcetone bath.To the chilled filtrates is then added 10.2 n1. of 2,6-lutidine and thenethylchloroformate (8.7 ml.) W61 a five-minute period after which theresulting soluion of mixed anhydride is stirred in the ice bath for 30ninutes. An anhydrous solution of 16.8 gm. of sodium iydrosulfidetrihydrate in 150 ml. of dimethylformamide s mixed with the mixedanhydride solution over a period )f five minutes and the resultingslurry is stirred for one mm at 0 C. The slurry is then slowly decantedinto 1500 ml. of ice water having a pH of 1.9. The pH of the nixture ismaintained at about 2 by adjustment With 6 N iydrochloric acid. Theaqueous solution of 6-[D,L-a- Jhenoxypropionamido]thiopenicillanic acidis extracted 1nd re-extracted with trichlorethylene. The combined:richl-orethylene extracts are dried over anhydrous magnesium sulfate inan ice bath and placed under vacuum for 30 minutes to remove hydrogensulfide. The magnesium sulfate is then filtered off and washed on thefilter with trichlorethylene. To the combined filtrates is added 70 ml.of a 22% solution of potassium ethylhexanoate in methyl isobutyl ketonewhereupon the product, potassium 6- [D,L-ot-phenoxypropionamido]thiopenicillanate, crystallizes out of solution. The slurry is stirredfor 20 minutes at room temperature and thereafter in an ice bath for 75minutes While the product crystallizes. The product is collected byfiltration, washed with trichlorethylene, vacuum dried over P 0 for 18hours and thereafter found to weigh 23.9 gm.

' Preparation 22.Preparati0n of potassium 6- [D,L-u

phenoxybutyramido] thiopenicillanate Potassium 6-[D,L-ot-phenoxybutyramido] penicillanate (30.0 gm.) is dissolved in amixture of 100 ml. ice Water and 100 ml. trichlorethylene. After coolingthe mixture to 5 C., 6 N hydrochloric acid is added with stirring untila pH of 2 is attained. The trichlorethylene phase is separated andmaintained at 5 C. The aqueous phase is extracted again with 50 ml. oftrichlorethylene and the trichlorethylene extracts are combined, mixedwith 40 gm. of anhydrous magnesium sulfate and stirred in an ice bathfor 30 minutes. The combined extracts are then filtered, and the filtercake is washed with ml. trichlorethylene. The filtrates are placedtogether with 250 ml. dimethylformamide in a one-liter 3-necked flaskwith a drying tube vent and cooled to -3 C. in an ice-acetone bath. Tothe chiiled filtrates is then added 10.2 ml. 2,6-lutidine and thenethylchloroformate, 8.7 ml. over a five-minute period after which theresulting solution of mixed anhydride is stirred in the ice bath for 30minutes. An anhydrous solution of 16.8 gm. of sodium hydrosulfidetrihydrate in 150 ml. of dimethylformamide is mixed with the mixedanhydride solution over a period of five minutes and the resultingslurry is stirred for one hour at 0 C. The slurry is then slowlydecanted into 1500 ml. of ice water having a pH of 1.9. The pH of themixture. is maintained at about two by adjustment with 6 N; hydrochloricacid. The aqueous solution of 6-[D,L-a-phenoxybutyramido]thiopenicillanic acid is extracted with trichlorethylene. The combinedtrichlorethylene extracts are dried over anhydrous magnesium sulfate onan ice bath and placed under vacuum for 30 minutes to remove hydrogensulfide. The magnesium sulfate is then filtered 01f and washed on thefilter with trichlorethylene. To the combined filtrates is added ml. ofa 22% solution of potassium ethylhexanoate in methyl isobutyl ketonewhereupon the product, potassium 6-[D,L-ot-phenoxybutyramido]thiopenicillanate, crystallizes out of solution. The slurry is stirred for 20minutes at room temperature and thereafter in an ice bath for minutesWhile the product crystallizes. The product is collected by filtration,washed with trichlorethylene, vacuum-dried over P 0 for 18 hours, andthereafter the structure is confirmed by nuclear magnetic resonance andinfrared absorption data.

Preparation 23.-Preparati0n of 6-aminothiopenicillanic acid Afermentation broth is prepared by the fermentation of Escherichia coliunder submerged aerobic conditions according to conventional proceduresand found to contain 5,270 penicillin amidase units per ml.

A solution of 2.5 gm. calcium nitrate dihydrate dissolved in 6 ml. ofwater is added to one liter of such fermentation broth. After mixing,the broth is filtered, and the filtered mat washed with 150 ml. water.The filtered mat is then suspended in 200 ml. of water to which is added5 ml. of toluene, and the suspension is stirred for three hours. Afterstirring, the suspension is filtered and the collected solid materialswashed with ml. of water. The filtrate is stirred with 1 gm. activatedcarbon (Darco KB) and 1.2 ml. of Quaternary Ammonium Salt Mixture No. I.Quaternary Ammonium Salt Mixture No. I is commercially available fromArmour & Company of Chicago, Ill., under the trademark of Arquad 16-50and is a liquid quaternary ammonium salt mixture containing, by weight,about 45% hexadecyltrirnethylammonium chloride, about 3%octadecyltrimethylammonium chloride, about 2%octadecenyltrimethylammonium chloride, about 35% isopropanol, about 14%water, and about 1% sodium chloride.

After thorough mixing, all solid materials are remove from the mixtureby filtration, and there is obtained 280 ml. of an essentially pureaqueous solution of penicillin amidase assaying 1950 penicillin amidaseunits per ml. and containing about 10% of the penicillin amidaseoriginally present in the broth.

Benzylthiopenicillin (8 gm.) is prepared by the proce-' dure describedin United States Patent No. 2,751,378 and added to 400 ml. of penicillinamidase solution, prepared according to the procedure described above.This suspension is maintained at pH 8.0 and 35 C. for four hours, duringwhich time the enzyme brings about the enzymatic hydrolysis of thebenzylthiopenicillin. The benzylthiopencillin and phenylacetate(produced during the hydrolysis) are removed at pH 2 and 5 C. withmethyl isobutyl ketone. After extraction, a portion of the liquor (250ml.) is adjusted to pH 7 with sodium hydroxide and vacuum concentratedto 20 ml. The concentrate is adjusted to pH 4 with hydrochloric acid (6N) and cooled to 5 C. and allowed to stand for 20 hours at 5 C. duringwhich time crystallization occurs. The crystals are filtered, washedwith water (10 ml.) and then dry acetone. The 6-aminothiopenicillanicacid is recovered, weighs 0.29 gm. and the presence of the o H -CSHgroup and ,B-lactam ring is confirmed by infrared analysis.

Preparation 24.Preparali0n 0 o-(a-aminophenylacetamido)-zhi0penicillanic acid 6 (acarbobenzyloxyaminophenylacetamido)penicillanic acid which is obtainedby the reaction of equivalent quantities of 6- a-aminophenylacetamidopenicillanic acid and benzyl chlorocarbonate in aqueous sodium hy-

1. A COMPOUND OF THE FORMULA